The discovery of induced Pluripotent Stem Cells (iPSCs) by S. Yamanaka1,2, and very rapid progress in iPSC technology have opened up a new avenue in autologous regenerative medicine, whereby patient-specific pluripotent cells could potentially be derived from adult somatic cells. iPSCs have been reproducibly obtained in different cell types by forced expression of the OCT4, SOX2, c-MYC and KLF4 transcription factor cocktail or by an alternative combination of factors, substituting KLF4 and c-MYC by NANOG and LIN283.
Cellular senescence is linked to physiological aging, and is characterized by a stable cell cycle arrest in response to various forms of stress stimuli, including oncogene activation or extremely shortened telomeres called replicative senescence9,10. A common feature is the activation of the p53/p21CIP1 and pRb/p16INK4A tumor suppressor pathways in these cells, associated with alteration of morphology, increase in senescence-associated β-galactosidase (SA-β-Gal) activity, a specific SA secretome (SASP) and formation of senescence-associated heterochromatic foci (SAHF), which are thought to be involved in repression of genes that promote cell division11.
EP 2 096 169 discloses a process for generating induced pluripotent stem cells from somatic cells, comprising the step of introducing the following six genes: Oct family gene, Klf family gene, Sox family gene, Myc family gene, Lin28 and Nanog into somatic cells. However, this specific combination of reprogramming factors has never been applied to senescent cells or cells from aged donors.
It has been recently described by several groups, that cellular senescence is a barrier to reprogramming, due to up-regulation of p53, p16INK4A, and p21CIP1, suggesting that cellular aging might be an important limitation of this technology. Accordingly, ablation of different senescence effectors has been proposed as a potential solution to improve the efficiency of iPSCs generation4-8.
WO 2011/016588 suggests using functional inhibitors of p53 together with a cocktail of reprogramming factors consisting of Oct3/4, Sox2, Klf4, L-myc and Lin28. p53 shRNA were used as functional inhibitors of p53.
The inventors have now shown that using the specific combination of the six factors OCT4, NANOG, SOX2, KLF4, c-MYC and LIN28 allows efficient reprogramming of both proliferative centenarian and senescent fibroblasts into human iPSCs, without the need of ablating senescence effectors, contrary to the technical prejudice in the art related to reprogramming of senescent cells.
Moreover, the inventors showed that this reprogramming restores telomere size, gene expression profile, oxidative stress and mitochondrial metabolism as observed in human embryonic stem cells (hESCs). Surprisingly, iPSCs derived from aged and senescent cells do not retain detectable marks of the cellular aging phenotype, and are indistinguishable from hESCs. Finally, iPSCs re-differentiated into fibroblasts exhibit an increased potential to proliferate, and gene expression profile equivalent to young proliferative fibroblasts, demonstrating that the reprogramming strategy according to the present invention erases the hallmarks of the cellular aging phenotype, defining a new method to produce rejuvenated cells.
To the applicant's knowledge, the invention is the first description of a method for producing iPSCs with cells from aged donors or senescent cells, therefore the invention may be highly useful in particular in autologous regenerative medicine, whereby patient-specific pluripotent cells could potentially be derived from adult aged or senescent somatic cells, and will also find numerous applications in the research field. Moreover, the invention is useful as a general method to rejuvenating senescent cells or cells from aged donors, either in vitro or in vivo.